Protocol for Construction of a Tunable CRISPR Interference (tCRISPRi) Strain for Escherichia coli.

We present a protocol for construction of tunable CRISPR interference (tCRISPRi) strains for Escherichia coli. The tCRISPRi system alleviates most of the known problems of plasmid-based expression methods, and can be immediately used to construct libraries of sgRNAs that can complement the Keio collection by targeting both essential and nonessential genes. Most importantly from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step oligo recombineering. Additional advantages of tCRISPRi over other existing CRISPRi methods include: (1) tCRISPRi shows significantly less than 10% leaky repression; (2) tCRISPRi uses a tunable arabinose operon promoter and modifications in transporter genes to allow a wide dynamic range with graded control by arabinose inducer; (3) tCRISPRi is plasmid free and the entire system is integrated into the chromosome; (4) tCRISPRi strains show desirable physiological properties.

tCRISPRi: tunable and reversible, one-step control of gene expression.

The ability to control the level of gene expression is a major quest in biology. A widely used approach employs deletion of a nonessential gene of interest (knockout), or multi-step recombineering to move a gene of interest under a repressible promoter (knockdown). However, these genetic methods are laborious, and limited for quantitative study. Here, we report a tunable CRISPR-cas system, "tCRISPRi", for precise and continuous titration of gene expression by more than 30-fold. Our tCRISPRi system employs various previous advancements into a single strain: (1) We constructed a new strain containing a tunable arabinose operon promoter PBAD to quantitatively control the expression of CRISPR-(d)Cas protein over two orders of magnitude in a plasmid-free system. (2) tCRISPRi is reversible, and gene expression is repressed under knockdown conditions. (3) tCRISPRi shows significantly less than 10% leaky expression. (4) Most important from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step of oligo recombineering. Our results show that tCRISPRi, in combination with recombineering, provides a simple and easy-to-implement tool for gene expression control, and is ideally suited for construction of both individual strains and high-throughput tunable knockdown libraries.

[Simulation of TDLAS direct absorption based on HITRAN database].

Simulating of the direct absorption TDLAS spectrum can help to comprehend the process of the absorbing and understand the influence on the absorption signal with each physical parameter. Firstly, the basic theory and algorithm of direct absorption TDLAS is studied and analyzed thoroughly, through giving the expressions and calculating steps of parameters based on Lambert-Beer's law, such as line intensity, absorption cross sections, concentration, line shape and gas total partition functions. The process of direct absorption TDLAS is simulated using MATLAB programs based on HITRAN spectra database, with which the absorptions under a certain temperature, pressure, concentration and other conditions were calculated, Water vapor is selected as the target gas, the absorptions of which under every line shapes were simulated. The results were compared with that of the commercial simulation software, Hitran-PC, which showed that, the deviation under Lorentz line shape is less than 0. 5%, and that under Gauss line shape is less than 2. 5%, while under Voigt line shape it is less than 1%. It verified that the algorithm and results of this work are correct and accurate. The absorption of H2O in v2 + v3 band under different pressure and temperature is also simulated. In low pressure range, the Doppler broadening dominant, so the line width changes little with varied.pressure, while the line peak increases with rising pressure. In high pressure range, the collision broadening dominant, so the line width changes wider with increasing pressure, while the line peak approaches to a constant value with rising pressure. And finally, the temperature correction curve in atmosphere detection is also given. The results of this work offer the reference and instruction for the application of TDLAS direct absorption.

Positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in Escherichia coli.

The two-step process of selection and counter-selection is a standard way to enable genetic modification and engineering of bacterial genomes using homologous recombination methods. The tetA and sacB genes are contained in a DNA cassette and confer a novel dual counter-selection system. Expression of tetA confers bacterial resistance to tetracycline (Tc(R)) and also causes sensitivity to the lipophillic chelator fusaric acid; sacB causes sensitivity to sucrose. These two genes are introduced as a joint DNA cassette into Escherichia coli by selection for Tc(R). A medium containing both fusaric acid and sucrose has been developed, in which, coexpression of tetA-sacB is orders of magnitude more sensitive as a counter-selection agent than either gene alone. In conjunction with the homologous recombination methods of recombineering and P1 transduction, this powerful system has been used to select changes in the bacterial genome that cannot be directly detected by other counter-selection systems.

Bacterial DNA polymerases participate in oligonucleotide recombination.

Synthetic single-strand oligonucleotides (oligos) with homology to genomic DNA have proved to be highly effective for constructing designed mutations in targeted genomes, a process referred to as recombineering. The cellular functions important for this type of homologous recombination have yet to be determined. Towards this end, we have identified Escherichia coli functions that process the recombining oligo and affect bacteriophage λ Red-mediated oligo recombination. To determine the nature of oligo processing during recombination, each oligo contained multiple nucleotide changes: a single base change allowing recombinant selection, and silent changes serving as genetic markers to determine the extent of oligo processing during the recombination. Such oligos were often not incorporated into the host chromosome intact; many were partially degraded in the process of recombination. The position and number of these silent nucleotide changes within the oligo strongly affect both oligo processing and recombination frequency. Exonucleases, especially those associated with DNA Polymerases I and III, affect inheritance of the silent nucleotide changes in the oligos. We demonstrate for the first time that the major DNA polymerases (Pol I and Pol III) and DNA ligase are directly involved with oligo recombination.

Probing cellular processes with oligo-mediated recombination and using the knowledge gained to optimize recombineering.

Recombination with single-strand DNA oligonucleotides (oligos) in Escherichia coli is an efficient and rapid way to modify replicons in vivo. The generation of nucleotide alteration by oligo recombination provides novel assays for studying cellular processes. Single-strand exonucleases inhibit oligo recombination, and recombination is increased by mutating all four known exonucleases. Increasing oligo concentration or adding nonspecific carrier oligo titrates out the exonucleases. In a model for oligo recombination, λ Beta protein anneals the oligo to complementary single-strand DNA at the replication fork. Mismatches are created, and the methyl-directed mismatch repair (MMR) system acts to eliminate the mismatches inhibiting recombination. Three ways to evade MMR through oligo design include, in addition to the desired change (1) a C·C mismatch 6 bp from that change; (2) four or more adjacent mismatches; or (3) mismatches at four or more consecutive wobble positions. The latter proves useful for making high-frequency changes that alter only the target amino acid sequence and even allows modification of essential genes. Efficient uptake of DNA is important for oligo-mediated recombination. Uptake of oligos or plasmids is dependent on media and is 10,000-fold reduced for cells grown in minimal versus rich medium. Genomewide engineering technologies utilizing recombineering will benefit from both optimized recombination frequencies and a greater understanding of how biological processes such as DNA replication and cell division impact recombinants formed at multiple chromosomal loci. Recombination events at multiple loci in individual cells are described here.

The involvement of replication in single stranded oligonucleotide-mediated gene repair.

Targeted gene repair mediated by single-stranded oligonucleotides (SSOs) has great potential for use in functional genomic studies and gene therapy. Genetic changes have been created using this approach in a number of prokaryotic and eukaryotic systems, including mouse embryonic stem cells. However, the underlying mechanisms remain to be fully established. In one of the current models, the 'annealing-integration' model, the SSO anneals to its target locus at the replication fork, serving as a primer for subsequent DNA synthesis mediated by the host replication machinery. Using a lambda-Red recombination-based system in the bacterium Escherichia coli, we systematically examined several fundamental premises that form the mechanistic basis of this model. Our results provide direct evidence strongly suggesting that SSO-mediated gene repair is mechanistically linked to the process of DNA replication, and most likely involves a replication intermediate. These findings will help guide future experiments involving SSO-mediated gene repair in mammalian and prokaryotic cells, and suggest several mechanisms by which the efficiencies may be reliably and substantially increased.

Identification of factors influencing strand bias in oligonucleotide-mediated recombination in Escherichia coli.

Recombinogenic engineering methodology, also known as recombineering, utilizes homologous recombination to create targeted changes in cellular DNA with great specificity and flexibility. In Escherichia coli, the Red recombination system from bacteriophage lambda has been used successfully to modify both plasmid and chromosomal DNA in a highly efficient manner, using either a linear double-stranded DNA fragment or a synthetic single-stranded oligonucleotide (SSO). The current model for Red/SSO-mediated recombination involves the SSO first annealing to a transient, single-stranded region of DNA before being incorporated into the chromosome or plasmid target. It has been observed previously, in both eukaryotes and prokaryotes, that mutations in the two strands of the DNA double helix are 'corrected' by complementary SSOs with differing efficiencies. Here we investigate further the factors that influence the strand bias as well as the overall efficiency of Red/SSO-mediated recombination in E.coli. We show that the direction of DNA replication and the nature of the SSO-encoded mismatch are the main factors dictating the recombinational strand bias. However, the influence that the SSO-encoded mismatch exerts upon the recombinational strand bias is abolished in E.coli strains that are defective in mismatch repair (MMR). This reflects the fact that different base-base mispairs are corrected by the mutS/H/L-dependent MMR pathway with differing efficiencies. Furthermore, our data indicate that transcription has negligible influence on the strand bias. These results demonstrate for the first time that the interplay between DNA replication and MMR has a major effect on the efficiency and strand bias of Red/SSO-mediated recombination in E.coli.